Interpretive Summary: The Hessian fly is a destructive pest of wheat, and in the eastern soft-winter-wheat region of the United States it is the most important insect pest. Losses in Georgia due to the Hessian fly were estimated at $28 million in 1989, and nation-wide monetary losses due to the pest have approached $100 million in a single year. Resistant wheat is the most economical and environmentally sound method of control. However, the widespread use of resistant wheat has resulted in the selection of biotypes of Hessian fly that can survive on formerly resistant wheat. The use of combinations (pyramids) of resistance genes has been proposed to be an effective way to increase the durability of insect-resistant crops. Naturally occurring inhibitors in plants of the protein digesting enzymes (proteases) in the guts of insects are attractive for their potential use in developing insect-resistant plants. We have cloned the two major protein digesting enzymes in the guts of Hessian fly larvae. This will allow us to test the effectiveness of plant inhibitors of these digestive enzymes for developing Hessian fly-resistant wheat. The genes for these inhibitors can then be combined through genetic engineering with native Hessian fly resistance genes in wheat to develop more durable resistance. Scientists facing the challenge of devising innovative methods of pest control through genetic engineering and other contemporary approaches will benefit from this fundamental knowledge. The agricultural community (crop producers and commodity groups) will benefit from improved pest control that increases yield and quality without increasing costs.

Technical Abstract: The Hessian fly, Mayetiola destructor, is a destructive pest of wheat and occurs in all production areas in the United States. Genetically resistant wheat is the most economical and environmentally sound method of control. However, the widespread use of resistant cultivars has resulted in the selection of biotypes that can survive on formally resistant wheat. Previous work has shown the major digestive proteolytic activity in midguts of M. destructor larvae is chymotrypsin-like and is strongly inhibited by the Bowman-Birk inhibitor. In the present work, two full-length cDNAs encoding chymotrypsin-like and trypsin-like proteases, respectively, have been cloned and characterized. The deduced amino acid sequence for the M. destructor serine proteases showed 63'72% similarity with trypsin-like and chymotrypsin-like proteases from Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, and Culex pipiens. Transcription profiling by real-time RT-PCR revealed the transcript for the chymotrypsin-like protease was predominant in feeding larvae. These results should allow use of RNAi silencing to test compensation between the two midgut digestive proteases, the effect of suppressing expression of digestive proteases on the development of M. destructor larvae, and the efficacy of protease inhibitors to develop M. destructor-resistant wheat via genetic engineering.